BIOL114 - Lecture 6

1.To generate large amounts of specific proteins at low cost with high purity. 2.) Where appropriate, to enable these proteins to be modified in ways that improve their properties = protein engineering.

1 of 70

Why might this be important?

Because many proteins are expressed at very low levels. Availablity of the the source tissue may be very limited. Source tissue can potentially be contaminated.

2 of 70

Give an example of a therapeutic protein:

Human insulin - it is much better to produce a human recombinant treatment then use animals.

3 of 70

Give 4 examples of recombinant therapeutic proteins:

Insulin, Human growth hormone, Factor viii, tPA

4 of 70

How can recombinant proteins be expressed in E.coli?

1.) Expression vectors (plasmids designed for transcribing and translating a cloned cDNA insert.

It has the necessary sequences that allow the inserted cDNA sequence to be both transcribed and translated. So transcription is driven by the promoter region and there is a ribosome binding site where translation occurs.

9 of 70

A cDNA which you've taken from a Eukaryotic cell will not have

a ribosomal binding site.

10 of 70

The expression vector also has a... because this is not found on the cDNA.

Terminator of transcription

11 of 70

In order for an expression vector to work, what must be the case:

Inserted DNA must be cDNA (no introns), Inserted cDNA must be adjacent to appropriate sequences for its transcription and translation in the host cell. Must be a high copy number plasmid.

12 of 70

What are the methods for improving levels of expression?

Inducible promoter?

13 of 70

What does inducible mean?

You can switch it on when you want it on.

14 of 70

Why would you want an inducible promoter?

Allow the expression of the recombinant protein to be switched on only when the bacterial culture has become established.

15 of 70

Why are inducible promoters advantageous?

Because recombinant proteins are often toxic for the bacteria and slow frowth - so there is strong selection pressure for elimination and rearrangement of the plasmid.

16 of 70

Which inducible promoter is most frequently used?

lac promoter - regulates transcription of the lac operon

17 of 70

How does the lac operon work?

When lactose is absent, the repressor is active and lac operon is off., so no RNA is made.

18 of 70

What happens when lactose is present?

The repressor is inactive, lac operon is on and therefore mRNA is made.

19 of 70

What is IPTG?

A stable analogue of lactose which binds to the Lac repressor protein. It is an effective inducer.. Lac repressor IPTG complex no longer binds to lac operator. Addition of IPTG to culture induces expression from the lac promoter.

20 of 70

What are the methods of improving expression?

Optimising codon usage. There are distinct idfference between codon usage in E.coli and humans that are refleted in the abundance of tRNAs, making translation of human sequences inefficient.

21 of 70

Codon optimization strategies -

Chemically synthesize new gene - alter sequence of the gene of interest to match donor codons to the codons most frequently used in host organisms.

22 of 70

How many codon optimization strategies are there?

2

23 of 70

What are the two codon optimization strategies?

Chemically synthesize new gene and use and engineered host cell that over expresses low abundance tRNAs

24 of 70

Why are N terminal fusions good?

They allow higher expression levels (rates of translation), increase stability of expressed protein - particularly if proteins are small, also used to facilitate purification of the protein - fusion tags.

25 of 70

The expression should have:

Protease cleavage site to allow the N terminal fusion to be removed later if necessary, multiple cloning site to facilitate insertion of the cDNA encoding the protein of interest.

26 of 70

After what can the N terminal be removed?

After affinity purification it can be removed if it is linked to the rest of the protein by a suitable protease target sequence e.g. thrombin.

To be stable at 70'C, resistant to non-ionic detergents, resistant to oxidation (for longer shelf life)

46 of 70

What is Psedomonas syringae?

It is a bacterium that colonises plant surfaces. Some strains contain a membrane proein known as ice nucleation protein (INP) that acts as a template for the formation of ice crystals.

47 of 70

How can microorganisms be used to convert one organic compound into another?

Synthesis of theraputic steroids from diosgenin.

48 of 70

What is bioremediation?

The process of reclaiming or cleaning up contaminated sites using microorganisms to remove or degrate toxic wastes. Used in sewage treatment, chemical degradation in soils, bioremediation of oil spills.

49 of 70

What happens during the bioremediation of oil spills?

Spraying of fertilizer to stimulate growth of indigenous bacteria that can breakdown the oil - fastest and cheapest way to clean up beaches.

50 of 70

What are three elements needed adjacent to a cDNA for expression in a bacterial plasmid?

Promoter, transcription terminator, Ribosome binding sequence.

51 of 70

What are three therapeutic recombinant proteins?

Human insulin, Erythropoietin, tPA

52 of 70

What are three reasons for expressing recombinant proteins as fusion proteins?

Increased expression, simpler purification, greater stability

53 of 70

What are 3 fusion tags?

Polyhistidine, Glutathione-S-transferase, Maltose Binding protein

54 of 70

What are three ligans used in purifying fusion proteins?

Nickel, Amylose, Gluthathione

55 of 70

What is a protein sequence used to direct protein secretion?

Signal peptide

56 of 70

What is an enzyme used in biological washing powder?

Subtilisin

57 of 70

What is an Ice-nucleating bacterium?

Pseudomonas syringae

58 of 70

What is a precursor for steroid production by microbial biotransformation?

Diosgenin

59 of 70

Explain why cDNA is ued to express eukaryotic proteins in bacteria

f

60 of 70

List the features of typical bacterial expression vectors

f

61 of 70

List examples of medically proteins epressed in E.coli

J

62 of 70

Describe how insulin is produced from E.coli

f

63 of 70

Explain the various reasons why foreign proteins may be epressed as fusion proteins in E.coli

j

64 of 70

Describe the features of vectors used for expression of fusion proteins

f

65 of 70

List different fusion tags and their uses

d

66 of 70

List the characteristics of recombinant proteins that may be improved by protein engineering